Bottom Line:
Previous studies showed porf-2 functions as a modulator in central nerve system development.We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs.By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function.

ABSTRACTNeural stem cell (NSC) proliferation and differentiation play a pivotal role in the development of brain, the plasticity of the brain network, and the repair for brain function in CNS diseases. The mechanisms regulating NSC behavior are not well elucidated. Previous studies showed porf-2 functions as a modulator in central nerve system development. We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs. We also demonstrate that porf-2 inhibits the proliferation of NSCs in vivo and in vitro, but has no effect on NSC differentiation. We investigated which domain is required for the role of porf-2 on NSC proliferation. By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function. In addition, we surveyed a few classical pathways on NSC proliferation and found that porf-2 inhibits NSC proliferation by suppressing the β-catenin nuclear translocation. Taken together, we show for the first time that porf-2 inhibits NSC proliferation through Wnt/β-catenin pathway by its GAP domain.

Figure 3: Porf-2 inhibits NSC proliferation in vitro. (A) Confirming the overexpression of porf-2 protein by immunoblot. (B) The neurosphere morphology in control and porf-2 overexpression group. Scale bar: 50 μm. (C) Quantification of the diameter of neurosphere in each group. Data are mean ± SEM (n = 3). (D) Representative image of Edu positive cells in each group. The total NSC number was counted by DAPI. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 100 μm. Data are mean ± SEM (n = 4). *P < 0.05.

Mentions:
To further demonstrate the role of porf-2 on NSC proliferation, we overexpressed porf-2 in NSCs. The overexpression of porf-2 was confirmed by WB (Figure 3A). By using the neurosphere assay, we measured the diameter of NSC sphere and found that overexpression of porf-2 can significantly reduce the diameter of NSC sphere compared to control (Figures 3B,C). In the Edu assay, as shown in Figure 3D, ~34% NSCs in control and about 28% NSCs in porf-2 overexpression group were Edu positive, confirming that overexpression of porf-2 decreased NSC proliferation. Altogether, our results suggest that overexpression of porf-2 inhibits NSC proliferation in vitro.

Figure 3: Porf-2 inhibits NSC proliferation in vitro. (A) Confirming the overexpression of porf-2 protein by immunoblot. (B) The neurosphere morphology in control and porf-2 overexpression group. Scale bar: 50 μm. (C) Quantification of the diameter of neurosphere in each group. Data are mean ± SEM (n = 3). (D) Representative image of Edu positive cells in each group. The total NSC number was counted by DAPI. Quantification of the percentage of Edu positive cell number in each group. Scale bar: 100 μm. Data are mean ± SEM (n = 4). *P < 0.05.

Mentions:
To further demonstrate the role of porf-2 on NSC proliferation, we overexpressed porf-2 in NSCs. The overexpression of porf-2 was confirmed by WB (Figure 3A). By using the neurosphere assay, we measured the diameter of NSC sphere and found that overexpression of porf-2 can significantly reduce the diameter of NSC sphere compared to control (Figures 3B,C). In the Edu assay, as shown in Figure 3D, ~34% NSCs in control and about 28% NSCs in porf-2 overexpression group were Edu positive, confirming that overexpression of porf-2 decreased NSC proliferation. Altogether, our results suggest that overexpression of porf-2 inhibits NSC proliferation in vitro.

Bottom Line:
Previous studies showed porf-2 functions as a modulator in central nerve system development.We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs.By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function.

ABSTRACTNeural stem cell (NSC) proliferation and differentiation play a pivotal role in the development of brain, the plasticity of the brain network, and the repair for brain function in CNS diseases. The mechanisms regulating NSC behavior are not well elucidated. Previous studies showed porf-2 functions as a modulator in central nerve system development. We here show that porf-2, a conserved family of RhoGAPs, is highly and specifically expressed in NSCs. We also demonstrate that porf-2 inhibits the proliferation of NSCs in vivo and in vitro, but has no effect on NSC differentiation. We investigated which domain is required for the role of porf-2 on NSC proliferation. By using neurosphere formation and Edu assay we confirmed the GAP domain is necessary for its function. In addition, we surveyed a few classical pathways on NSC proliferation and found that porf-2 inhibits NSC proliferation by suppressing the β-catenin nuclear translocation. Taken together, we show for the first time that porf-2 inhibits NSC proliferation through Wnt/β-catenin pathway by its GAP domain.